The long-term goal of this proposal is to study the role which Ca2+ channels controlling neurotransmitter release play in chemical-induced toxicity, and disease processes. These channels are crucial to the cell signalling process. They are postulated to be target sites for chemicals of environmental interest such as Pb2+, Hg2+ and hydrazines as well as therapeutic agents such as general anesthetics and anticonvulsants. These channels may also be target sites in certain diseases. Lambert-Eaton Myasthenic Syndrome (LEMS) is one such disorder. It is an autoimmune disorder characterized by decreased evoked quantal release of acetylcholine (ACh) and disruption of the presynaptic active zones, the sites at which ACh is thought to be released. It is hypothesized that LEMS antibodies are directed fairly specifically at the Ca2+ channels located at or near the active zone. The initial goal of this project is to study the proposed site of action of the LEMS auto-antibody on nerve terminals directly in hopes of identifying and characterizing the Ca2+ channel subtype involved in the disease. The specific hypothesis to be tested is that acute and chronic exposure of nerve terminals isolated from rat brain to serum or IgG obtained from patients with LEMS impairs flux of Ca2+ through voltage- dependent Ca2+ channels during depolarization, and subsequent release of transmitter. For chronic studies, osmotic pumps and i.c.v. administration of LEMS serum/IgG to rats implanted with cannula in the lateral cerebral ventricles, will be used to attempt to induce a passive transfer of LEMS to Ca2+ channels in the central nervous system. Specificity of LEMS IgG for cholinergic axon terminals will be assessed by comparing the ability of LEMS IgG to block release of [3H]-ACh with release of [3H]-dopamine from enriched subtypes of cholinergic and monoaminergic synaptosomes respectively while effects of LEMS IgG on peripheral cholinergic terminals will be examined using synaptosomes derived from myenteric plexus. The potential role of early components of the Complement cascade, particularly C3, in the action of LEMS IgG will be assessed. The pharmacological subtype(s) of Ca2+ channel(s) affected by LEMS autoantibody will be characterized using binding of ligands thought to bind at distinct types of Ca2+ channels and electrophysiological analyses of whole cell and single Ca2+ channel currents. Synaptosomes will also be used to test whether LEMS sera/IgG reduces the number of Ca2+ channels in synaptosomes. Voltage clamp recordings will be made from Ca2+ channels expressed in Xenopus oocytes by injection of rat brain poly A(+) mRNA and from single synaptosomal membrane Ca2+ channels incorporated into bilayer membranes. Amplitudes of currents carried by divalent cations in the nerve terminal (as measured extracellularly) will be compared using in vitro neuromuscular preparations of normal rats and those treated intraperitoneally with LEMS IgG. The results of this proposal should provide direct evidence for or against the hypothesis that nerve membrane Ca2+ channels are a target site for auto-antibodies in LEMS. Results may also serve to identify an antibody specific for Ca2+ channels involved in transmitter release.